1. Field of the Invention
The present invention relates generally to the fields of enzymology, protein structure and drug screening. More specifically, the present invention relates to the use of a crystalline structure of an aldose reductase complexed with NADPH and glutathione conjugate as a screening tool for inhibitors of aldose reductase.
2. Description of the Related Art
Aldose reductase (AR) is a monomeric (a/b8-barrel (TIM barrel) protein belonging to the aldo-keto reductase (AKR) superfamily (1-3). Aldose reductase is a broad-specificity oxidoreductase catalyzing the reduction of a structurally-diverse range of aldehydes, including medium to long chain aldehydes, glucose and other aldo-sugars, aldehyde metabolites of neurotransmitters, isocorticosteroid hormones, and a variety of xenobiotic aldehydes to their corresponding alcohols (4). Reduction of glucose to sorbitol by aldose reductase constitutes the first and rate-limiting step of the polyol pathway that converts glucose to fructose via sorbitol dehydrogenase. Although this pathway usually represents a minor route of glucose metabolism, its activation during diabetes has been linked to the development of several clinically significant secondary complications such as nephropathy, neuropathy, retinopathy and cardiovascular related complications (4, 5). Several drugs that inhibit aldose reductase have been shown to prevent hyperglycemia-induced changes in nerve, kidney, and lens of experimental animals, although clinical trials with Type I and Type II diabetics have not been uniformly positive (4-6).
In addition to glucose, it has been shown that aldose reductase catalyzes the reduction, of multiple biologically-active aldehydes generated by the peroxidation of membrane lipids and lipoproteins (7-9) or during glucose (10) and amine (11) metabolism. The aldehyde-detoxifying role of aldose reductase is supported by the observation that inhibition of the enzyme increases the accumulation of lipid peroxidation products (12, 13) that cause cytotoxicity (14, 15). The most abundant and toxic lipid peroxidation product is 4-hydroxy-trans-2-nonenal (16) which is efficiently reduced by aldose reductase in vitro and in vivo.
A primary role of aldose reductase in aldehyde detoxification is consistent with its structure. The active site of the enzyme is highly hydrophobic and contains few polar residues typically required for binding sugars with high specificity and affinity (2, 3). These features are, however, compatible with binding to hydrophobic lipid-derived aldehydes. Additionally, the substrate-specificity of aldose reductase is unusually broad, in part because the enzyme derives most of the energy required to achieve a substrate transition state from cofactor-binding (17). The active site environment exerts low stabilization on the transition state (18). Furthermore, it has been demonstrated recently that aldose reductase-catalyzed products mediate cytokine, chemokine, growth factor, and hyperglycemia-induced signaling that activates NF-kB and AP1, and regulates vascular epithelial cell (VEC) and human lens epithelial cell (HLEC) apoptosis, and vascular smooth muscle cell (VSMC) proliferation (FIG. 1) (15, 21, 22).
The range of aldehydes recognized by the aldose reductase active site is increased further by the ability of the enzyme to bind glutathione-aldehyde conjugates (19, 20), such as glutathionyl HNE. Given the high concentration of reduced glutathione in most cells and the highly electrophilic nature of several aldose reductase substrates, it is possible that reduction of aldehyde-glutathione conjugates, in addition to free aldehydes, may be a primary in vivo function of aldose reductase and that glucose may be an incidental substrate of the enzyme. Previous kinetic studies showed that glutathiolation increases the catalytic efficiency with which unsaturated aldehydes are reduced by aldose reductase (19), suggesting that the active site of aldose reductase contains a specific glutathione-binding domain (20). Nevertheless, the precise nature of glutathione binding to aldose reductase remained unclear.
There is a need in the art for three-dimensional structures of aldose reductase-glutathione-moiety binding complexes to understand the nature of glutathione-moiety binding at the active site. Also there is a need for methods incorporating computer modeling of three-dimensional structures to identify, design and test molecules with improved binding affinity. A further need for molecules that would be useful as therapeutics and/or modulators of aldose reductase-mediated physiological events is also present in the art.
The prior art is deficient in structure based aldose reductase inhibitors that preferentially occlude one binding site in the inhibitor. Specifically, the prior art is deficient in the lack of aldose reductase:NADPH:glutathione-like ligand based inhibitors that inhibit binding and reduction of glutathione-lipid aldehyde conjugates without inhibiting the detoxification of free aldehydes. The present invention fulfills this long-standing need and desire in the art.